Ral proteins are members of the Ras family of small GTPases that participate in cell proliferation, differentiation, endocytosis, and regulation of the actin cytoskeleton. Ral proteins can be found in caveoli of the plasma membrane, but most are present in endocytic and exocytic vesicles. They are activated by upstream Ral guanine nucleotide exchange factors (RalGEFs), which are most frequently activated by Ras. The overall goal of this proposal is to better understand the biological role of the Ral proteins. Four specific aims are proposed: 1) Dr. Feig's laboratory has recently found that Ral is an intermediary in EGF-activation of c-Src but not in isoproterenol-induced activation of c-Src. In this system, EGF/Ral-GTPase stimulation of c-Src results in phosphorylation of the c-Src substrates, STAT3 and cortactin, while the downstream signaling molecules which become tyrosine phosphorylated following isoproterenol stimulation include Shc and MAP kinase. Dr. Feig interprets these data to suggest that EGF/Ral regulate activation of c-Src and substrate specificity. He proposes to investigate other signaling pathways known to activate c-Src for Ral involvement and for their influence on c-Src signaling specificity. These include the PDGF, integrins, glutamate, estrogen and calcium-induced activations. 2) Dissect the mechanisms underlying the regulation of c-Src activity by Ral-GTPase. Preliminary data suggest that the Ral target protein, filamin, exists in a complex with c-Src. This relationship will be further investigated by determining (among other things) whether filamin is required for Ral-induced c-Src activation, which domains of filamin and c-Src participate in complex formation, and whether effector domain mutants of Ral affect activation of c-Src and selection of substrate specificity. 3) Study the regulation of Ral activation by Ral-GEFs. Ras-independent mechanisms of activating Ral-GEFs will be explored, including activation by PKC and the characterization of a newly discovered Ral-GEF (AND-34 or BCAR3) that is not regulated by Ras. 4) Investigate the possible role of Ral proteins in mammary carcinogenesis. Dr. Feig's laboratory has found that BCAR3 , a protein isolated previously on the basis of its ability to convert tamoxifen-dependent breast cancer cells to tamoxifen-independence, is a Ral-GEF. Therefore, he plans to determine if Ral plays a role in conversion of breast cancer cells to tamoxifen-independence, as well as other mechanisms of tumor progression.